Method for controlling hydrocarbon fermentations and apparatus therefor

ABSTRACT

THE CONCENTRATION OF A MODERATELY VOLATILE HYDROCARBON SUBSTRATE IN A FERMENTATION MEDIUM CAN BE READILY DETERMINED AND SAID CONCENTRATION MAINTAINED AT A PREDETERMINED LEVEL BY MONITORING THE VAPOR CONCENTRATION OF SAID HYDROCARBON IN THE FERMENTATION EXHAUST GASES AND CONTROLLING THE INTRODUCTION OF ADDITIONAL SUBSTRATE INTO THE FERMENTATION MEDIUM BASED ON THE AMOUNT OF SAID VAPOR CONTENT. THIS CAN ADVANTAGEOUSLY BE CARRIED OUT IN A CONTINUOUS MANNER BY AUTOMATED INSTRUMENTATION.

June 2 1971 P. HOSLER 3,586,605

METHOD FOR CONTROLLING HYDROCARBON FERMENTATIONS AND APPARATUS THEREFORFiled June 18, 1968 MONITOR ANALYZER,

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PETER HOSLER JOMJJK ATTORNEY United States Patent O1 fice Patented June22, 1971 US. Cl. 195-28 6 Claims ABSTRACT OF THE DISCLOSURE Theconcentration of a moderately volatile hydrocarbon substrate in afermentation medium can be readily determined and said concentrationmaintained at a predetermined level by monitoring the vaporconcentration of said hydrocarbon in the fermentation exhaust gases andcontrolling the introduction of additional substrate into thefermentation medium based on the amount of said vapor content. This canadvantageously be carried out in a continuous manner by automatedinstrumentation.

BACKGROUND OF THE INVENTION This invention relates to an improved methodfor controlling the concentration of hydrocarbon substrates infermentation media where said hydrocarbons are being oxidized bymicroorganisms to form oxygenated hydrocarbon derivatives. It alsorelates to an apparatus for achieving this purpose.

The microbiological oxidation of hydrocarbons to form oxidative productsthereof has been investigated and reported in numerous recent literatureand patent references. Not as well known or understood, however, is theneed for controlling the concentration of the hydrocarbon substrate inthe fermentation medium in order to achieve improved yields of thedesired oxidation products. Thus, in US. Pat. No. 3,458,399, filed bythe present applicant on Aug. 31, 1966, there is taught the desirabilityof controlling the rate of addition of the hydrocarbon feed so that theamount thereof in the dispersion in excess of the amount absorbed by themicroorganism cells does not exceed the limit of solubility of thehydrocarbon in the aqueous nutrient medium. This thus-desired control,as taught by this earlier application, was achieved by taking samples ofthe fermentation medium from time to time, testing them to determine theconcentration of hydrocarbon substrate in the aqueous phase of themedium and regulating the introduction of additional feed accordingly.While the effect of this sampling and control was to increase the yieldof oxidative product, nevertheless complex testing proceduress involvingcentrifuging the samples, extracting them with suitable solvents andanalyzing the extract by UV spectroscopy were required at each samplinginterval. Despite these elaborate methods, such steps were onlypartially successful in achieveing the desired control.

Therefore, if control of the hydrocarbon concentration were ever to beachieved on a practical basis, it became evident that the known samplingmethods would have to be improved upon in order to provide moresimplified and accurate methods for obtaining this desired objective.

Accordingly, it is an object of this invention to provide an improvedmethod, and apparatus, for closely and accurately measuring andcontrolling the concentration of a hydrocarbon substrate at apredetermined level in a fermentation medium where said hydrocarbon isundergoing microbiological oxidation. This and other objects of thepresent invention will be apparent from the following description.

SUMMARY OF THE INVENTION In accordance with the present invention it hasnow been found that the concentration of a moderately volatilehydrocarbon substrate in an aqueous fermentation medium can be readilydetermined and said concentration closely controlled and maintained at apredetermined level by:

(1) Establishing under aerobic fermentation conditions an aqueousfermentation medium comprising a hydrocarbon-oxidizing microorganism anda predetermined amount of a liquid hydrocarbon substrate dispersed in anaqueous nutrient medium;

(2) Sampling the exhaust gas from said fermentation to determine thevapor content of said hydrocarbon in said exhaust gas; and

(3) Controlling the introduction of additional hydrocarbon substrateinto the fermentation medium in dependence upon the vapor content ofsaid hydrocarbon in the exhaust gas in order to maintain saidpredetermined concentration.

DESCRIPTION OF THE DRAWING The figure is a schematic view of a fermentorin combination with hydrocarbon vapor analyzer which actuates a feedpump for regulating the rate of introduction of hydrocarbon feedstuifinto the fermentor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As taught in US. Pat. No.3,458,399 (supra), in order to obtain maximum yields of oxygenatedhydrocarbon product from the fermentation of hydrocarbon feedstuifs, theintroduction of the feed must be carefully controlled to maintain itsconcentration in the aqueous phase below that corresponding to the limitof its solubility in the nutrient medium; the amount of hydrocarbonabsorbed by the cells of the microorganism is not counted for purpose ofthis determination. This solubility limit will depend mainly on theparticular hydrocarbon used and the fermentation temperature employed.Hydrocarbon solubility limits in the nutrient medium usually are lessthan 200 ppm. and often may be less than p.p.m. In some cases it may bebest to maintain a quite low hydrocarbon concentration, e.g., 5-20 ppm.as some microorganisms can be sensitive to and adversely affected by ahigher concentration even though it be substantially below thissolubility limit. Below a concentration of about 5 ppm, however, therate of fermentation is generally undesirably slow.

Inv oxidizing alkylbenzenes of the C C range, for example, best resultsare generally obtained when the unconsumed hydrocarbon concentration ismaintained at a level of about 5150 ppm. in the aqueous nutrient phase.For other types of substrates, and for any given microorganism, thisoptimum level can conveniently be determined experimentally by varyingthe hydrocarbon concentration levels and observing both the productyield and rate at which it is produced, as well as monitoring the oxygenand carbon dioxide levels in the exhaust gas of the fermentor. A.decrease in carbon dioxide and an increase in oxygen, for example, isindicative of an oversupply of hydrocarbon feed. The reverse is truewhen the feed supply is inadequate.

Once the Optimum concentration for any given combination of substrateand microorganism had been determined, it had been the practice formerlyto maintain this concentration by taking samples of the fermentationbroth at given intervals, separating the cells from those samples bycentrifugation or filtration, extracting the unconsumed hydrocarbon,determining the amount extracted, and adding additional feed inaccordance with this determination.

It has now been found, however, in accordance with the presentinvention, that these time-consuming and laborious steps can be avoided,and the concentration of hydrocarbon in the fermentation brothcontrolled much more closely and accurately, by directly measuring thevapor content of the hydrocarbon feedstuif in the exhaust gases of thefermentor without having to resort to taking samples of the fermentationbroth. That is to say, it has been found that the concentration ofhydrocarbon in the exhaust gas is directly proportional to theconcentration of hydrocarbon in the aqueous fermentation. There thusexists an equilibrium distribution of the hydrocarbon between theaqueous fermentation and the fermentation exhaust gas which convenientlypermits the monitoring of the hydrocarbon vapor content of the exhaustgas by appropriate measuring devices. Preferably, continuous monitoringby automatic instruments is provided Where the signal from the exhaustgas analyzer may then be used to determine the hydrocarbon feed rate,desirably by actuating a feed mechanism.

While applicant does not wish to be bound by any particular theory, thedirect correlation that has been found to exist between the vaporcontent of the hydrocarbon in the exhaust gas and its concentration inthe aqueous phase of the fermentation broth is apparently based on theprinciple that the partial vapor pressure of any volatile constitutentof a solution is equal to the vapor pressure of the pure constituentmultiplied by the mol fraction of that constituent in solution. Based onthis principle the concentration in the aqueous phase can readily bedetermined from the vapor content and the rate of feed to thefermentation broth conveniently controlled and maintained by appropriatedevices.

The limits of the relationship between the vapor phase content and theaqueous phase content of the hydrocarbon are the solubility ofhydrocarbon in water, and the saturation of the exhaust air stream, asinfluenced by the partial pressure of the hydrocarbon at fermentationtemperature. In the case of a fermentation of xylene, for example, wherethe xylene is fermented at 30 C., the distribution has been found tofollow the following relationship: 1 mg. xylene/liter of water=0.84 mg.xylene/liter of exhaust as. g The limit of the above relationship isapproximately 37 mg. of xylene per liter of exhaust gas, which is thesaturation point.

While various modes of operation will be apparent to those skilled inthe art based on the foregoing description of the invention, the figurehas been included to illustrate one typical method of operation which isapplicable to different combinations of microorganisms and hydrocarbonsubstrates.

In the figure there is shown fermentor containing culture medium 11which has been inoculated with a suitable microorganism. The fermentoris fitted with agitator 12 driven by motor 13. The agitator may be oneor more in number depending upon the degree of agitation and aerationdesired for any given fermentation. Fermentor 10 is also fitted with anaerator 14 through which sterile air can be bubbled from pipe 15.Optionally the fermentor 4 may be fitted with a pH measuring devicewhich actuates a container of alkali, in order to maintain a given pHlevel.

At the top of the fermentor 10 the gases are exhausted with pressurefrom the pressurized fermentor through pipe 16 fitted with aback-pressure regulating valve 17. These gases are then passed alongpipe 16 to which are attached oxygen monitor 18, carbon dioxide monitor19, and a hydrocarbon analyzer 20. The remaining unsampled gases arethen vented from pipe 16.

The oxygen and carbon dioxide monitors, which may be of any conventionalconstruction, are employed for the purpose described earlier ofmeasuring the respiratory activity of the cells, which in turn is ameasure of their product-forming ability. While monitors 18 and 19 arenot essential to the performance of this invention, they do simplify theoverall control of the fermentation system, and particularly thestart-up procedures.

The electrical signal from hydrocarbon analyzer 20 is used to activatepneumatic converter 21, which may be of any conventional design. Theregulated air output of this converter is, in turn, used to activatepneumatic valve 22. The hydrocarbon feed, in a pressurized vessel 23, isthus supplied through pipe 24 to the fermentor 10 in a proportional modeof operation to maintain a predetermined concentration of hydrocarbonsubstrate dispersed in the aqueous medium. If it is desired to maintainasepsis of operation, the hydrocarbon can be sterlized by passing itthrough a steam-jacketed heat exchanger 25. The start-up of thisoperation is ordinarily done manually to avoid initial overshoot untilthe overall system has reached equilibrium by responding to the vaporcontent of the exhaust gas.

As an alternative to proportional control the hydrocarbon feedstuif canbe pumped to the fermentor using a simple relay to provide ofiF-oncontrol of a pump attached to vessel 23, which relay is activated by theoutput of the hydrocarbon analyzer. This mode of control can be quiteeffective if care is taken to set the pumping rate only slightly inexcess of required feed rate, so that excessive over-supply ofhydrocarbon is avoided.

The hydrocarbon analyzer may be one of several different types which arecommercially available. Thus, for example, a unit which measuresultra-violet light at the particular wavelength of the particularhydrocarbon feedstuff is suitable for purposes of this invention,especially if the hydrocarbon feed is an aromatic compound.Alternatively, a total hydrocarbon analyzer with a flame ionizationdetector may be employed instead, particularly if the hydrocarbon is astraight chain alkane.

The present invention is applicable to a wide variety of processesinvolving the fermentation of hydrocarbonoxidizing microorganisms incombination with various hydrocarbon substrates to produce manydifferent types of oxygenated hydrocarbon products. Microorganisms wh1chmay be employed in these fermentations include bacteria, yeasts, moldsand actino mycetes, and particularly such genera as Micrococcus.Corynebacterium, Nocardia, Pseudomanas, Mycobacterium, Streptomyces,Aspergillus, and Acetobacter.

The hydrocarbon substrates include any moderately volatile, normallyliquid hydrocarbon having from 5 to 12 carbon atoms, and particularlythe aromatic hydrocarbons such as the C C benzenoid hydrocarbons. Thus,for example, such hydrocarbons as benzene, toluene, xylene, hexane,octane, decane, dodecane and biphenyl or the like may suitably beemployed in this process.

Among the types of oxidations wherein this invention has particularutility are in the oxidations of alkyl groups of alkylbenzenes asdescribed in US. Pat. 3,057,784. The invention is likewise applicable tothe oxidation of alkylnaphthalenes such as l-methylnaphthalene,l-ethylnaphthalene and the like, as well as to normally liquid alkanesof the C -C range, particularly n-parafiins and cycloparafiins.

Except for the control of the introduction of the liquid hydrocarbonsubstrate into the fermentation medium, the fermentation may be carriedout in a conventional fashion wherein a nutrient medium is introducedinto a fermentor provided with means for aeration and agitation, themedium inoculated with a suitable microorganism, and the fermentation isthen effected by continually adding the hydrocarbon feed at a properrate while maintaining fermentation conditions. Generally a temperatureof from -40 C. is suitable, and the pH should be maintained in the rangeof 4-9, and preferably 6-8. The hydro carbon substrate may be added fromthe beginning, or else the cells can be grown on another substrate andthen the hydrocarbon feed added.

After maximum accumulation of the oxygenated product, the fermentationbroth can be treated in any suitable manner to recover the product. Forexample, the product can be extracted directly from the Whole broth bymeans of a suitable solvent, or else the cells may be first removed bycentrifugation or filtration, followed by processing of the clear broth.

The nutrient medium used in the process should contain sources ofavailable nitrogen, phosphorous, sulfur and magnesium and may containvarious trace elements and vitamins as conventionally employed or asrequired by the particular microorganism being used. Mineral saltscustomarily used for supplying such elements in biological fermentationscan be employed. Examples of suitable nitrogen sources are ammoniumsalts such as (NH SO or NH Cl, nitrate salts such as NH NO or NaNO urea,soybean meal and other organic nitrogen sources. The followingillustrates a suitable mineral salt composition for the present purpose:

Conc., g./1. of H 0 0.2 Na CO 0.1 CaCl 2H O 0.01 MnSO -H O 0.02 FeSO 7HO 0.005 Na HPO 3 .0 KH PO 2.0 Urea 2.0

This mineral salt composition normally would have a pH of about 7.1.When it is desired to carry out the fermentation at a pH below 7, theamount of KH PO relative toNa HPQ, can be increased to reduce the pH toa lower 4 Nocardia corallina ATCC No. 19,070 was used to preparedimethyl muconic acid (DMMA) from p-xylene in a 760 liter fermentor ofthe type shown in FIG. 1, together with an automatic pH monitoringsystem. A mineral salt solution of the following composition was used asthe aqueous nutrient medium:

Conc., g./l. of H 0 MgSO -7H O 0.2 N11200:; 0.1 CaCl 2H O 0.01 MnSO -H O0.02 FeSO -7H O 0.005 Na HPO 3.0 KH PO 2.0 Urea 2.0

520 liters of the solution were charged and sterilized and the mixturewas inoculated with the organism. The mixture was stirred vigorously at30 C. while being aerated at a constant rate of 2 c.f.m. Normalparafiins Were used as a growth substrate and were added at the rate ofml./hr. Also the pH level was automatically held at 6.8 during the runby addition of aqueous caustic soda using a pH controller. The exhaustgas was monitored by an ultraviolet absorption instrument, and thecontrol apparatus was set to maintain 7 mg. xylene per liter of exhaustgas. At the end of the fermentation at about 50 hours analysis of thewhole broth showed that it contained about 15.4 gm. of DMMA per liter.

EXAMPLE II In this run Nocardia: salmonicolor ATCC 19,149 was used tooxidize p-xylene to dihydroxy p-toluic acid (DHPT) and p-toluic acid(PTA). The procedure and apparatus were the same as in Example I. 34liters of a mineral salt mixture were sterilized in a 60 liter fermentorprovided with aeration and agitation. The mixture was inoculated withthe organism and stirred vigorously at 30 C. while being aerated at aconstant rate of 9 liters per minute. Normal paraflins were used as agrowth substrate and were added at the rate of 7 ml./hour. At 12 hoursthe cell mass had increased to approximately 3 gm. dry weight per liter.At this time the pH was raised from about 7 to pH 8, and the xylenemaintained at 20 gm. per liter of exhaust gas. After 20 hours of thecontrolled xylene addition the fermentation broth was analyzed and foundto contain 4.2 gm. of DHPT and 5.3 gm. of PTA per liter.

EXAMPLE III The culture Nocardia salmonicolor ATCC 19,149 was grown onbenzene as a growth carbon source to provide quantities of cell mass ofthe culture. 34 liters of a mineral salt mixture were sterilized in a 60liter fermentor provided with aeration and agitation. The mixture wasinoculated with the organism and stirred vigorously at 30 C. while beingaerated at a constant rate of 6 liters per minute. Benzene was added tothe system described above in Example I to maintain 12 mg. benzene perliter of exhaust gas. After 25 hours of operation growth was determinedby centrifugation at 3600 rpm. A packed cell volume of 3% was obtained.

What is claimed is:

1. A process for determining the concentration, in an aqueousfermentation medium, of a hydrocarbon having from 5 to 12 carbon atoms,and maintaining said concentration at a predetermined level whichcomprises (a) establishing under aerobic fermentation conditions anaqueous fermentation medium;

(b) sampling the exhaust gas from said fermentation to determine thevapor content of said hydrocarbon in said exhaust gas; and

(c) controlling the introduction of additional hydrocarbons into thefermentation medium in dependence upon the vapor content of saidhydrocarbon in said exhaust gas in order to maintain said predeterminedconcentration.

2. The process according to claim 1 wherein the sampling and controllingare carried out in a continuous manner.

3. The process according to claim 1 wherein the sampling and controllingare carried out automatically.

4. The process according to claim 1 in which the hydrocarbon is analkylaromatic compound having from 5 to 12 carbon atoms.

5. The process according to claim 1 in which the hydrocarbon is anormally liquid alkane having from 5 to 10 carbon atoms.

6. A fermentation system for the controlled fermentation of hydrocarbonshaving from 5 to 12 carbon atoms wherein said hydrocarbons aremaintained in a fermentation medium at predetermined concentrationswhich comprises, in combination, a fermentor containing a suitablefermentation medium, said fermentor being equipped with means forintroducing controlled amounts of hydrocarbons into said fermentationmedium and means for aeration and agitation of said medium, means forremoving exhaust gases from the upper end of said fermentor, ahydrocarbon analyzer adapted to be receptive to the hydrocarbon vaporcontent of said exhaust gases, said analyzer being constructed andarranged to produce an output corresponding to said hydrocarbon vaporcontent, and means responsive to said output for controlling the outputof the hydrocarbon-introducing means in amounts proportional to theoutput of said analyzer in order to maintain said hydrocarbons atpredetermined concentration in the fermentation medium.

References Cited UNITED STATES PATENTS 3,010,881 11/1961 Markhof 195133X3,383,289 5/1968 Raymond et al 195-28 3,384,553 5/1968 Caslavsky et a1.19594X 10 A. LOUIS MONACELL, Primary Examiner S. RAND, AssistantExaminer US. Cl. X.R.

